Assay systems for determining the concentration of one or more analytes in a test sample with a high degree of accuracy are frequently required. These systems find a wide scope of application ranging from the determination of toxic substances in industrial wastes to the determination of potential contaminants in food supplies. The development of assay systems for analytes in biological fluids, such as serum, plasma, urine and the like, has received much attention due to the need of physicians for accurate, up-to-date information concerning the physiological condition of their patients to assist in diagnostic and therapeutic activities. As a result, assay systems capable of determining the concentration of various analytes in biological fluids with a high degree of accuracy have evolved.
One such system is the well-known radioimmunoassay in which a radiolabel conjugated to an analog of the analyte or an antibody is employed. In its classical form, a known amount of a radiolabeled analog of the analyte is allowed to compete with the analyte for a limited quantity of antibody specific for the analyte. In other forms, the radiolabel may be conjugated to an analyte-specific antibody, which forms a "sandwich" with analyte present in the test sample. Without regard to test format, the radiolabel complexed with the analyte or remaining free in solution is measured as an indication relating either directly or indirectly to the amount of analyte in the test sample. Although highly sensitive radiolabel-based assay systems have been developed, the requirement of radioactive materials, specialized handling procedures and specialized equipment, and the production of radioactive wastes present serious drawbacks to widespread, continuing use of radiolabel-based assays.
The requirement for radioactive materials in the assay art has been decreased by the use of enzyme label materials in place of radiolabels. Typical enzyme immunoassays, for example, follow assay protocols very similar to those employed in corresponding radioimmunoassays, with the amount of enzyme activity, determined colormetrically, varying directly or indirectly with the amount of analyte in a test sample. Widespread use of enzyme labelling systems has significantly reduced the amount of radioactive materials which would otherwise have been employed for clinical assay purposes, but has frequently resulted in loss of assay sensitivity, high background levels and equivocal assay results.
More recently, luminescent indicator reactions have been proposed for use in place of radiolabels or enzyme labels in otherwise conventional assay systems. Luminescent reactions, based on the measurement of light emitted by an assay system component, have been investigated for replacement of radioactive and enzyme labels in immunoassay formats and as a replacement for conventional colorimetric or spectrophotometric indicator reactions, such as in assays for substrates of oxidases and hydrolases. The art has generally recognized two types of luminescent indicator reactions useful in assay systems-fluorescent reactions and chemiluminescent reactions. In fluorescent assay systems, a luminescent molecule is promoted to an excited energy state by the transfer of energy from incident radiation produced by a light source to the luminescent molecule. The molecule thereafter relaxes to a lower energy state with the emission of light in a manner and amount dependent on the amount of the luminescent molecule present in the system. In chemiluminescent assay systems, however, no incident radiation is required. Chemiluminescence is broadly defined as the energy product of reacting two or more chemical reactants to obtain an electronically excited, energy-rich reaction product which relaxes to a lower energy state (e.g., ground state) with the emission of light. Methods for the determination of analytes in solution using chemiluminescent techniques are now well established in the art. Examples of patents describing various chemiluminescent assay systems are set forth below.
U.S. Pat. No. 3,689,391 of Ullman discloses the use of chemical or electrochemical reactions to yield molecules in their electronically excited, energy-rich states, and the transfer of energy from those molecules (energy donors) to reactant molecules (energy receptors) that then undergo photochemical transformation yielding emitted light.
U.S. Pat. No. 4,104,029 of Maier, Jr. discloses a procedure for the assay of biochemically active compounds in biological fluids by combining in an aqueous reaction mixture a medium suspected of containing the compound of interest, a chemiluminescent-labelled ligand and a soluble receptor having sites capable of bonding to the ligand and to the chemiluminescent-labelled ligand. The ligand and the chemiluminescent-labelled ligand then compete for bonding interaction with the ligand receptor (antibody). After equilibrium, the receptor (antibody) is isolated from the medium and measured for chemiluminescence, the amount of chemiluminescent-labelled ligand bound to the antibody being related to the amount of unlabelled ligand in the solution being assayed.
U.S. Pat. No. 4,160,645 of Ullman describes a catalyst-mediated competitive protein binding assay which utilizes a chemiluminescent label conjugated to a ligand analog, a first redox reactant which reacts by one-electron transfer, and a second redox reactant which reacts by two-electron transfer, by comparing the rate of reaction between the first and second redox reactants and the chemiluminescent label with the corresponding rate in an assay solution having a known amount of analyte.
U.S. Pat. No. 4,161,516 of Ullman discloses a double receptor fluorescent immunoassay employing a ligand analog conjugated to a fluorescer, an antibody to the ligand and an antibody to the fluorescer, wherein the amount of fluorescer bound to the antifluorescer is related to the amount of ligand present in an unknown sample and the difference in emission spectrum between unbound fluorescer and fluorescer bound to the antibody.
U.S. Pat. Nos. 4,220,450 and 4,277,437 of Maggio disclose a chemiluminescent competitive protein binding assay wherein a chemiluminescent label is conjugated to one member of an immunological pair, a quencher molecule is conjugated to the other member of an immunological pair and the amount of analyte present in an assay medium is determined by observing the light emitted from the medium.
U.S. Pat. No. 4,231,754 of Vogelhut discloses a multi-layer chemiluminescent test device including a solid carrier, a first layer having a first reagent system responsive to the presence of an analyte to produce a reaction product, and a second layer having a second reagent system responsive to the presence of the reaction product to produce luminescence. The second reagent system may include a cyclic hydrazide, such as luminol, a ferric ion, hemoglobin, hematin or microperoxidase product catalyst, and a buffer for maintaining the pH of the chemiluminescent reaction medium at from 8.5 to 12.5.
U.S. Pat. No. 4,238,195 of Boguslaski et al. discloses a specific binding assay for determining a ligand, such as an antigen or an antibody, by chemically exciting a fluorescent label and measuring light emitted by the label. The fluorescent label is chemically excited by exposure to a high energy intermediate such as hydrogen peroxide and either oxylochloride, an oxamide or a bis-oxylate ester.
U.S. Pat. No. 4,269,938 of Frank discloses a peroxidase assay conducted by contacting a sample with diacetyl dichlorofluorescin and a source of hydrogen peroxide to form a fluorescent product. When all reactants other than peroxidase are present in excess, the rate of fluorescence increase is related to the amount of peroxidase in the sample assayed.
U.S. Pat. No. 4,302,534 of Halmann et al. discloses a heterogeneous immunoassay in which chemiluminescence is produced by an enzymatic catalyzed redox reaction between hydrogen peroxide and a phenolic compound, such as pyrogallol, resorcinol, phloroglucinol or hydroquinone. In the assay, horseradish peroxidase, lactoperoxidase, turnip peroxidase or potato peroxidase is conjugated to an antibody or antigen, the conjugate is reacted with a sample, excess conjugate is removed, luminescent substrate is added and then light emitted from the system is measured.
U.S. Pat. No. 4,372,745 of Mandell et al. discloses a system for the detection of a biological analyte, including a microencapsulated fluorescer material conjugated to an immunological binding partner specific to the analyte, means for disrupting the capsule and an energy source other than electromagnetic radiation capable of activating a fluorescer.
Chemiluminescent assay systems, particularly those catalyzed by horseradish peroxidase, have been widely reported and are known to have a number of significant advantages over other conventional signal labels commonly used in the art, including relatively high sensitivity, low cost, extended linear range, relatively simple signal measuring equipment and the lack of use of radioactive isotopes, thereby eliminating the need for safety equipment and special handling procedures. Despite these advantages, the use of chemiluminescent assay systems has not been without problems. For example, peroxidase catalyzed oxidations of luminol are highly sensitive to changes in pH. Conventional horseradish peroxidase (HRP) ehemilumineseent luminol oxidations are typically conducted at a pH in the range of 8 to 12, with the most efficient chemiluminescence being produced at a pH of about 10.4 to 11.5. The HRP-catalyzed dioxygenation of luminol to produce chemiluminescence is a highly complex process, involving the reaction of HRP with hydrogen peroxide to form a first complex (Complex I), which in turn reacts with luminol to yield a second complex (Complex II) and an oxidized luminol radical. Complex II then reacts with a second luminol molecule to yield a second oxidized luminol radical and HRP in its initial state. In essence, two luminol molecules (2LH.sub.2) are dehydrogenated from the hydrazide to yield two luminol radicals (2LH.sup..) according to the reaction: ##STR1## The radicals are then believed to react with additional hydrogen peroxide to form aminophthalate with the release of nitrogen and emission of a photon (h.nu.) of light as follows: EQU 2LH.sup.. +H.sub.2 O.sub.2 .fwdarw.LH.sub.2 +aminophthalate+N.sub.2 +h.nu.
The luminescence reaction is best achieved when H.sub.2 O.sub.2 is present in its conjugate base form, HO.sub.2.sup.-. However, the relatively high pKa of H.sub.2 O.sub.2 (11.6; Lange's Handbook of Chemistry, 13th Edition, 1985) dictates a relatively high pH for HO.sub.2 to be present in significant amounts, as follows.
______________________________________ RATIO OF [HO.sub.2.sup.- ] TO [H.sub.2 O.sub.2 ] AT VARIABLE pH pH [HO.sub.2.sup.- ]/[H.sub.2 O.sub.2 ] ______________________________________ 12.65 10 11.65 1 10.65 10.sup.-1 9.65 10.sup.-2 8.65 10.sup.-3 7.65 10.sup.-4 6.65 10.sup.-5 5.65 10.sup.-6 4.65 10.sup.-7 3.65 10.sup.-8 ______________________________________
For example, the effective concentration of HO.sub.2.sup.- in a one millimolar solution of H.sub.2 O.sub.2 is one nanomolar at pH 5.65. In order to obtain efficient oxidation of luminol, it has therefore been a common practice to conduct HRP-catalyzed oxidation at alkaline pH levels, particularly at a pH of about 7 to 12, and more commonly at a pH of 9 to 11. At high pH levels, however, luminol undergoes base catalyzed oxidation even in the absence of a catalytic enzyme, resulting in peroxide consumption, high background chemiluminescence and frequently in unacceptably low signal-to-noise ratios.
These pH requirements pose serious limitations to the widespread use of peroxidase catalyzed luminol luminescence for clinical and research applications. For example, peroxidase catalysis is most efficient over the pH range of 7 to 9; above a pH of 9, peroxidase exhibits substantially lower activity. However, oxidase enzymatic reactions typically exhibit an optimum pH in the range of about 5 to about 7. When oxidase enzymatic reactions are employed to produce hydrogen peroxide for measurement by a peroxidase catalyzed indicator system, the primary enzymatic processes producing hydrogen peroxide cannot occur simultaneously with the luminescent reaction (where a significantly higher pH is optimum) without severe compromises to luminescent intensity and the rates of the enzyme-catalyzed reactions. In addition to increasing the background chemiluminescence, the relatively high pH levels required for the peroxidase catalyzed luminescent reaction may accelerate the rate of reaction between hydrogen peroxide and reducing components in the biological sample, thereby consuming hydrogen peroxide before it can react with luminol, decreasing the observed luminescence from the system and artificially interfering with accurate measurement of the arialyre of interest. See Seitz, W. R., "Chemiluminescence Detection of Enzymatically Generated Peroxide", Meth. Enzymol., 57: 445-462 (1978).
U.S. Pat. No. 4,598,044 of Kricka et al. discloses an enhanced luminescent reaction between a peroxidase enzyme, an oxidant and a 2,3-dihydro-1,4-phthalazinedione in which the total light emission from the luminescent reaction is stated to be increased (or the signal/noise ratio is enhanced) by adding certain phenolic compounds into the luminescent reaction mixture. The enhanced assay, however, is still preferably conducted at alkaline pH. The use of phenolic compounds to enhance to accelerate the peroxidase catalyzed oxidation of other lumiphores is also disclosed in U.S. Pat. No. 4,521,511. Although the use of enhancers has been shown to improve luminescent determinations, assay sensitivities and poor signal-to-noise ratios continue to prevent the widespread use of these luminescent systems in the clinical environment.
In the "enhanced" luminescent assay using phenolic enhancers to increase light emission, a further problem has been described with the storage stability of reagents used in the assay when the reagents are maintained at the high pH required for conducting the chemiluminescent reaction. To overcome this problem, European Patent Application Publication No. 235,970 describes maintaining the pH of the reagents in the range of about 3 to about 6 prior to use. However, in the assay system disclosed in this European application, an alkaline buffer must be used during the ehemilumineseent reaction to raise the pH of the reaction mixture to a value in the range of 7 to 9 to obtain efficient light emission.
As is apparent from the foregoing, the pH dependency of the light emitting reaction has significantly limited the usefulness of peroxidase catalyzed chemiluminescent techniques as assays in the past. A strong need exists for improved chemiluminescent indicator systems which will overcome problems and limitations associated with prior art ehemilumineseent assays.
In addition to the luminescent assay systems described above, chemical reactions which produce chemiluminescence have been known to occur naturally in various biological systems. For example, myeloperoxidase (MPO) is an oxidoreductase which makes up as much as 5% by weight of mammalian polymorphonuclear (PMN) leukocytes. The detoxification activity of MPO on diphtheria toxin in the presence of hydrogen peroxide was first described by Agner (Nature, Vol. 159, p. 271, 1947), as was its dependence on a halide cofactor (Agner, J. Exp. Med., Vol. 92, p. 334, 1950; Agner, Rec. trav. chim., Vol. 74, p. 373, 1955; Agner, Abstr. Communs. 4th Congr. Biochem. Vienna, p. 64, 1958). The antibacterial effect of MPO, a halide and an hydrogen peroxide on Escherichia coli or Lactobacillus acidophilus has been described by Klebanoff in "Myeloperoxidase-Halide-Hydrogen Peroxide Antibacterial System," J. Baeteciol., Vol. 95, pp. 2131-2138 (1968). The antibacterial activity of MPO in the MPO-haldine-hydrogen peroxide system is accompanied by a native (i.e., no chemiluminigenic substrate added) chemiluminescence (Allen,"Studies on the Generation of Electronic Excited States in Human Polymorphonuelear Leukoeytes and Their Participation in Microbiocidal Activity," Dissertation, Tulane University, Jul. 13, 1973), which is pH dependent (Allen, "The Role of pH in the Chemiluminescent Response of the Myeloperoxidase-Halide-HOOH Antimicrobial System," Biochem. and Biophys. Res. Comm., Vol. 63, No. 3, pp. 684-691, 1975) and halide dependent (Allen, "Halide Dependence of the Myeloperoxidase-Mediated Antimicrobial System of the Polymorphonuclear Leukocyte in the Phenomenon of Electronic Excitation," Biochem. and Biophys. Res. Comm., Vol. 63, No. 3, pp. 675-683, 1975).
The oxidoreductase eosinophil peroxidase (EPO) is present in high concentration in eosinophils and has been shown to have an antiparasitic function similar to that of MPO (Caulfield et al., J. Cell. Biol., Vol. 86, pp. 64-76, 1980). EPO catalyzes the oxidation of chloride ion to hypochlorous acid in the presence of hydrogen peroxide at acid pH (Ben et al., "Some Properties of Human Eosinophil Peroxidase, A Comparison With Other Peroxidases," Biochim. et Biophys. Acta, Vol. 784, pp. 177-186, 1984). Other chloroperoxidases are known and have been characterized in the literature, such as that isolated from the mold Caldariomyces fumago as described by P. D. Shaw and L. P. Hager (JACS, Vol. 81, No. 1001, p. 6527, 1959; JBC, Vol. 234, p. 2560, 1959, Vol. 234, p. 2565, 1960, and Vol. 236, p. 1626, 1961).
Although the in vivo native MPO/EPO-halide-HOOH antibacterial system (without added chemiluminigenic substrate) has been studied and reported in the literature, the use of a haloperoxidase-halide-oxidant-luminescent substrate indicator system for the determination of the presence or amount of an analyte in a sample has not been reported or suggested in the art.